Monoclonal antibody B72.3, which has been extensively characterized as to reactivity with a variety of carcinomas versus normal tissues, reacts with a mucin-like glycoprotein. The B72.3-reactive antigen, designated TAG (tumor-associated glycoprotein) -72, was partially purified and used as immunogen to produce 18second generation anti-TAG-72 antibodies. Based on competitive binding studies, a number of the epitopes recognized by the anti-TAG-72 antibodies appear to be structurally or spatially related, but none appear to be identical. All of the anti-TAG-72 antibodies produced immune precipitate lines with purified antigen in double immunodiffusion assays, indicating that each epitope recognized by the antibodies is expressed in multiple copies on the TAG-72 molecule. All of the epitopes are sensitive to mild periodate oxidation but differ in their sensitivities to neuraminidase, and all of the epitopes have been shown to be expressed on the same molecule or population of molecules. one of the second generation antibodies was selected to develop procedures to purify preparative amounts of TAG-72 from xenografted human colon carcinoma cells and from malignant effusions. TAG-72, extracted from xenografted human colon carcinoma cells, was subjected to antibody affinity chromatography, size exclusion chromatography, and ion exchange chromatography. A double determinant radioimmunoassay revealed a greater than 1000-fold purification with a greater than 10% yield. SDS-PAGE analysis of radiolabeled purified TAG-72 revealed a polydisperse, but apparently homogenous, high molecular weight glycoprotein. The density of purified TAG-72, as determined by equilibrium centrifugation in a CsCl gradient, was 1.45 g/ml, indicating a high degree of glycosylation. Compositional analysis of the purified TAG-72 revealed amino acid and carbohydrate profiles and content highly similar to that reported for other purified mucins. TAG-72 purified from malignant effusions was shown to have properties highly similar to the TAG-72 purified from the colon carcinoma xenografts. The colon carcinoma-associated antigen recognized by monoclonal antibody D612 has been shown to be a 48 kilodalton glycoprotein, consisting of a 42 kilodalton polypeptide and three N-linked oligosaccharides. The D612 antigen has also been shown to be structurally and antigenically unrelated to the colon carcinoma-associated antigens defined by monoclonal antibodies CO17-1A, GA733, KS1/4, etc.